TULANE COBRE: PHOSPHORYLATION IN SOLID MUSCLE TUMOR ALVEOLAR RHABDOMYOSARCOMA

TULANE COBRE：实性肌肉肿瘤肺泡横纹肌肉瘤的磷酸化

• 批准号: 6972569
• 负责人: ANDREW DURRELL HOLLENBACH
• 金额: $ 31.15万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-17 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6972569
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; chimeric proteins; chromosome translocation; genetic transcription; mass spectrometry; muscle neoplasms; myoblasts; myogenesis; phosphorylation; rhabdomyosarcoma; transcription factor; western blottings

The solid muscle tumor Alveolar Rhabdomyosarcoma (ARMS), one of the most frequent soft tissue sarcomas in children, is characterized by the t(2;13)(q35;q14) chromosomal translocation, which results in the fusion of two transcription factors important for muscle development, Pax3 and FKHR. At present, little is known about the underlying molecular mechanisms regulating the activities of Pax3 and the oncogenic fusion protein Pax3-FKHR and how this regulation may be important for the development of ARMS. Therefore, the overall objective of this proposal is to understand how phosphorylation regulates the transcriptional activity of Pax3 and how Pax3-FKHR may alter this regulation. This objective will be addressed through two specific aims: (1) To investigate the role of phosphorylation in the regulation of Pax3 and Pax3-FKHR. This specific aim will be addressed by identifying the sites of phosphorylation on Pax3 and Pax3-FKHR, determining how phosphorylation affects the molecular biological activities of each protein (i.e. - DNA-binding, protein stability, and transcriptional activity), determining how phosphorylation alters the Pax3- and Pax3-FKHR-dependent enhanced proliferation and differentiation of primary myoblasts, and analyzing the importance of phosphorylation on the oncogenic activity of Pax3-FKHR. (2) To identify the kinases responsible for phosphorylating Pax3 and Pax3-FKHR. This specific aim wil'. be addressed by using standard chromatographic techniques to isolate the kinases that phosphorylate Pax3 and Pax3-FKHR from primary myoblasts, confirming that these kinases are responsible for the phosphorylation in vivo, and utilizing kinase inhibitors to determine how inhibition of these kinases effects the previously described Pax3 and Pax3-FKHR-dependent biological effects (i.e. - enhanced proliferation, enhanced apoptosis, and oncogenic activity). The comparison of the differences in the regulation of Pax3 and Pax3-FKHR by phosphorylation will provide important insights into the role of Pax3-FKHR in the formation of ARMS.

以t(2；13)(q35；q14)染色体易位为特征的实体肌肉瘤--泡状横纹肌肉瘤(ARMS)是儿童最常见的软组织肉瘤之一，导致Pax3和FKHR两个转录因子的融合。目前，对Pax3和致癌融合蛋白Pax3-FKHR活性调节的潜在分子机制以及这种调节如何在ARM的发育中发挥重要作用还知之甚少。因此，本提案的总体目标是了解磷酸化如何调节Pax3的转录活性，以及Pax3-FKHR如何改变这一调节。这一目标将通过两个特定的目标来实现：(1)研究磷酸化在Pax3和Pax3-FKHR调节中的作用。这一具体目标将通过确定Pax3和Pax3上的磷酸化位点来实现 Pax3-FKHR，确定磷酸化如何影响每个蛋白质的分子生物学活性(即DNA结合、蛋白质稳定性和转录活性)，确定磷酸化如何改变依赖Pax3和Pax3-FKHR的原代成肌细胞的促进增殖和分化，以及分析磷酸化对Pax3-FKHR致癌活性的重要性。(2)鉴定参与Pax3和Pax3-FKHR磷酸化的蛋白激酶。这一具体目标将会实现。通过使用标准的层析技术从原代成肌细胞中分离磷酸化Pax3和Pax3-FKHR的激酶，证实这些激酶负责体内的磷酸化，并利用激酶抑制剂来确定抑制这些激酶如何影响先前描述的依赖于Pax3和Pax3-FKHR的生物学效应(即促进增殖、增强细胞凋亡和致癌活性)。比较Pax3和Pax3-FKHR在磷酸化调控方面的差异，将为了解Pax3-FKHR在ARM形成中的作用提供重要的见解。